New potential Plasmodium brasilianum hosts: tamarin and marmoset monkeys (family Callitrichidae).

BACKGROUND: Non-human primates (NHPs) as a source for Plasmodium infections in humans are a challenge for malaria elimination. In Brazil, two species of Plasmodium have been described infecting NHPs, Plasmodium brasilianum and Plasmodium simium. Both species are infective to man. Plasmodium brasilianum resembles morphologically, genetically and immunologically the human quartan Plasmodium malariae. Plasmodium brasilianum naturally infects species of non-human primates from all New World monkey families from a large geographic area. In the family Callitrichidae only the genus Saguinus has been described infected so far. The present study describes the natural infection of P. brasilianum in tamarins and marmosets of the genera Callithrix, Mico and Leontopithecus in the Atlantic forest. METHODS: One hundred and twenty-two NHPs of the family Callitrichidae housed in the Primate Centre of Rio de Janeiro (CPRJ) were sampled in June 2015, and January and July 2016. The CPRJ is located in the Atlantic forest in the Guapimirim municipality, in the Rio de Janeiro state, where human autochthonous cases of malaria have been reported. The samples were screened for the presence of Plasmodium using optical microscopy and nested PCR for detection of 18S small subunit rRNA gene. The amplicon was sequenced to confirm the molecular diagnosis. RESULTS: The frequency of Plasmodium infections detected by nested PCR in New World monkeys of the family Callitrichidae was 6.6%. For the first time, Callitrichidae primates of genera Callithrix, Mico and Leontopithecus were found naturally infected with P. brasilianum. Infection was confirmed by sequencing a small fragment of 18S rRNA gene, although no parasites were detected in blood smears. CONCLUSIONS: The reported P. brasilianum infection in NHP species maintained in captivity suggests that infection can be favoured by the presence of vectors and the proximity between known (and unknown) hosts of malaria. Thus, the list of potential malaria reservoirs needs to be further explored.

Improving N-terminal protein annotation of Plasmodium species based on signal peptide prediction of orthologous proteins.

BACKGROUND: Signal peptide is one of the most important motifs involved in protein trafficking and it ultimately influences protein function. Considering the expected functional conservation among orthologs it was hypothesized that divergence in signal peptides within orthologous groups is mainly due to N-terminal protein sequence misannotation. Thus, discrepancies in signal peptide prediction of orthologous proteins were used to identify misannotated proteins in five Plasmodium species. METHODS: Signal peptide (SignalP) and orthology (OrthoMCL) were combined in an innovative strategy to identify orthologous groups showing discrepancies in signal peptide prediction among their protein members (Mixed groups). In a comparative analysis, multiple alignments for each of these groups and gene models were visually inspected in search of misannotated proteins and, whenever possible, alternative gene models were proposed. Thresholds for signal peptide prediction parameters were also modified to reduce their impact as a possible source of discrepancy among orthologs. Validation of new gene models was based on RT-PCR (few examples) or on experimental evidence already published (ApiLoc). RESULTS: The rate of misannotated proteins was significantly higher in Mixed groups than in Positive or Negative groups, corroborating the proposed hypothesis. A total of 478 proteins were reannotated and change of signal peptide prediction from negative to positive was the most common. Reannotations triggered the conversion of almost 50% of all Mixed groups, which were further reduced by optimization of signal peptide prediction parameters. CONCLUSIONS: The methodological novelty proposed here combining orthology and signal peptide prediction proved to be an effective strategy for the identification of proteins showing wrongly N-terminal annotated sequences, and it might have an important impact in the available data for genome-wide searching of potential vaccine and drug targets and proteins involved in host/parasite interactions, as demonstrated for five Plasmodium species.